This proposal aims to define the mechanisms by which Staphylococcus epidermidis and S. aureus form both mono-species and mixed-species biofilms, focusing on the cell wall-anchored proteins Aap and SasG. This application builds on the previously funded R01 project in which we defined the structural basis for Zn2+- mediated dimerization of the adhesive B-repeat region of Aap and the determinants for stability of this unusual protein fold. We also showed that Aap contains two B-repeat subtypes with distinct assembly and stability characteristics, allowing us to decipher an `assembly code' for intercellular adhesion in staphylococcal biofilms. Importantly, we have demonstrated that Aap is a multi-functional adhesion protein. In its full-length, auto- inhibited form, it mediates adhesion to host cells, but after proteolytic processing, the inhibition is released and the intercellular adhesion region is unmasked. Aap is capable of two assembly modes: reversible oligomerization (similar to that observed in our crystal structures) and formation of amyloid-like fibrils that are resistant to environmental stresses. Furthermore, recent reports indicate that Aap is capable of heterophilic interactions with other biofilm proteins such as small basic protein (SBP) and the Aap ortholog from S. aureus, SasG. We have shown that S. epidermidis and S. aureus can form robust, synergistic mixed-species biofilms that have important implications for a number of disease states. The goal of this application is to broadly characterize the reversible self-assembly modes and heterophilic interactions involving Aap; the irreversible assembly of Aap into the functional amyloid state; and the mechanism for auto-inhibition that governs the switch between host attachment and intercellular adhesion in the nascent biofilm. We are collaborating with a leader in the field of staphylococcal genetics to express full-length Aap variants on the S. epidermidis cell surface. We will use these strains expressing Aap variants to explicitly test the relative contribution of reversible Aap assembly and amyloid fibril formation in biofilm growth, as well as the role of heterophilic assembly events involving SBP and SasG in the formation of mono-species and mixed-species biofilms. We will test each strain under low- and high-shear conditions in a new flow cell apparatus to mimic biofilms that form in blood vessels or catheters. Relevance: Healthcare-associated infections (HAIs) are a major cause of patient morbidity and mortality; a recent CDC report estimated that HAIs cause 75,000 deaths annually in the United States. Staphylococci are the most common infective agents in HAIs. The propensity of Staphylococci to form biofilms?specialized surface-adherent colonies that are resistant to antibiotics?leads to recurrent, hard-to-treat infections. The proposed research will provide insights into how staphylococcal cells are anchored to one another in the biofilm and aid in the development of targeted approaches for antimicrobial therapy.